Materials and Methods for Improving Immune Responses and Skin and/or Mucosal Barrier Functions

ABSTRACT

The subject invention provides compositions and methods for treating human dermatological conditions by employing a microbiome-centered treatment approach. Preferred embodiments of the invention provide pharmaceutical and cosmetic compositions, and the methods of using the same, comprising a strain of  Lactobacillus fermentum  bacterium, or bioactive extracts thereof, derived from human microbiota and capable of growing in biofilm phenotype.

CROSS-REFERENCE TO A RELATED APPLICATION

This application is a continuation application of U.S. application Ser.No. 15/496,686, filed Apr. 25, 2017; which is a continuation applicationof co-pending U.S. application Ser. No. 15/226,226, filed Aug. 2, 2016;which is a continuation of International Application No.PCT/US2016/042939, filed Jul. 19, 2016; which claims the benefit of U.S.Provisional Application Ser. No. 62/194,630, filed Jul. 20, 2015, all ofwhich are incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

With its diverse resident commensal microbiota, the human microbiome hasreceived substantial attention for the critical roles it plays in healthand disease.

An initial line of defense against foreign invaders, the skin is home toa diverse population of microbes. These microbes include residentcommensals, transients and pathogens. In order to survive in thechallenging environment of the skin, microbes often exhibit a biofilmphenotype, which gives competitive advantages for survival and growth.Many skin pathogens can be found living on the skin as commensals;microbial dysbiosis, host genetic variation, and immune status may drivethe transition from commensal to pathogen.

Atopic dermatitis (AD) is a multifactorial chronic inflammatory skindisease involving genetic factors, such as filaggrin deficiency (1), andenvironmental triggers, including Staphylococcus aureus, and is oftenthe initial presentation of the “allergic march.” Worldwide, AD affectsapproximately 20% of children and 5% of adults, typically presentingclinically as chronically dry, pruritic eczematous dermatitis withepisodic acute flares. AD is frequently associated with asthma andallergic rhinitis. Quality of life for individuals affected by AD issignificantly disrupted, and there is a heavy economic burden associatedwith the disease, with estimates of annual national direct costs rangingfrom 364M USD to 3.8B USD (4,5). AD represents a large, unmet need formore effective therapeutics.

Commensal bacteria like S. epidermidis have several antibacterialmechanisms to ward-off pathogenic bacteria. Several studies have shownserine proteins from commensal S. epidermidis prevent the growth ofpathogenic strains of S. aureus (40,41). A novel lipopeptide fromcommensal Staphylococcus epidermidis increases HβD2 and HβD3 viaTLR2/CD36-MAPK, thus enhancing antimicrobial defense against pathogenicinfections (42,43). Other studies have shown that S. epidermidis in thehuman skin microbiome produces secondary fermentation metabolites toinhibit the growth of additional pathogenic bacterial strains (44).Further, enhancing commensals such as S. epidermidis might have otherbeneficial roles, too. For example, skin S. epidermidis have anautonomous role in controlling the local inflammatory milieu and tuningresident T lymphocyte function, thereby rendering protective immunity toa cutaneous pathogen (45).

Skin dysbiosis has been linked with common skin conditions, includingAD, acne and rosacea (21). Skin microbiome sequencing during acute ADflares has correlated increased levels of the pathogen S. aureus and thecommensal S. epidermidis during flares, with subsequent decrease duringapplication of standard medical treatments such as topical steroids andantibiotics (22).

Skin dysbiosis can initiate key biochemical and immune triggers, andstudies have found an association between high amounts of staphylococcalbacteria and clinical worsening of AD lesions; for instance, patientsharboring MRSA had greater total body clinical dermatitis scores(15,29-31).

Lipoteichoic acid exerts immunological effects mainly through TLR 2(32-34) that could implicate it in the worsening of atopic dermatitis(31,35). Studies showed that in a pediatric population of AD patients,temporal shifts in the skin microbiota occur over three disease stages:baseline, flare, and post flare as compared to healthy controls. Inparticular, lesional skin bacterial diversity decreased during the flarestage, parallel with increased relative abundance of S. aureus, butincreased during the post flare status, indicative of a link betweendisease severity and microbial diversity (21,22).

Filaggrin deficiency, Staphylococcus aureus colonization, defectiveinnate immunity and skin microbial dysbiosis are the major underlyingfactors in the progression of the disease. Up to 90% of individuals withAD are colonized with S. aureus; prevalence of MRSA in AD ranges from10-30.8% (15).

The role of the skin barrier in the pathogenesis of AD is now clear(18-20). In this context, the strongest genetic association with AD sofar has been demonstrated for loss-of-function mutations in thefilaggrin (FLG) gene, which encodes the important barrier protein(pro-filaggrin) (8,9). Filaggrin plays several roles in thepathophysiology of AD which explains why lower expression of a singlecomponent of the epidermal differentiation complex might have such agreat influence on the whole function of the skin barrier. Skin barrierfunction is a major determinant of the equilibrium of skin commensalflora. The skin barrier protein FLG ensures that pathogenic strains ofbacteria are not penetrating into deeper layers. In a recent studyfilaggrin knockout resulted in significantly increased epidermal S.aureus colonization, as well as in an up-regulation of S. aureus-inducedIL-8 expression (16).

However, the FLG mutation is absent in most AD individuals; secondaryfilaggrin deficiency is common in AD (6). This has been postulated to bedue to various environmental factors, such as the Th2/Th22 cytokinemilieu common in AD inflammation, bacterial exotoxins, AD skin dysbiosisassociated with cutaneous S. aureus and methicillin-resistant S. aureus(MRSA), mechanical damage associated with scratching, skin dehydrationand topical irritants (9-14).

Lower filaggrin levels in AD predispose to S. aureus colonization(11,12) and lower levels of antimicrobial peptides are associated withdefective innate immunity and increased extracellular adhesins for S.aureus (16,17).

Controlling pathogenic biofilm and its metabolic products on skin is amajor component in restoring the symbiosis of commensal skin flora andskin health. Current therapeutics, however, focus mainly onsymptom-control rather than modulating the overall microbiome to inhibitthe growth and reduce the adhesion and attachment of pathogenic biofilmactivities, while promoting the growth of commensal biofilm on humanskin.

SUMMARY OF THE INVENTION

The subject invention provides compositions and methods for treatinghuman dermatological (and other) conditions by employing amicrobiome-centered treatment approach. Preferred embodiments of theinvention provide pharmaceutical and cosmetic compositions, and methodsof using the same.

In preferred embodiments, the compositions comprise a strain ofLactobacillus fermentum bacterium, or a bioactive extract thereof. Inpreferred embodiments, extracts of the bacteria are obtained when thebacteria are grown as biofilm. The subject invention also providescompositions comprising L. fermentum bacterium, or bioactive extractsthereof, in a lyophilized, freeze dried, and/or lysate form.

In some embodiments of the subject invention the enhancement of skinbarrier functions is achieved by upregulating the expression of skinbarrier proteins utilizing the bacterial compositions of the subjectinvention. In other embodiments, the enhancement of skin innate immunefunctions is provided by modulating the expression of skin innate immunepeptides and/or inflammatory cytokines utilizing the bacteria andcompositions of the subject invention.

In another aspect, the subject invention provides a method of treatinghuman dermatological disorders, comprising administering to a subject atherapeutically effective amount of the composition, wherein thecomposition preferably comprises one or more bioactive extracts of theLf Qi6 biofilm.

Advantageously, preferred compositions and treatment methods providedherein are effective in treating human skin barrier dysfunctions,dysbiosis, skin innate immune dysfunctions, and/or inflammatory skindiseases, as well as improving the appearance and/or the texture of theskin. Subjects treated according to the subject invention can experiencerelieved symptom severity, reduced incidence of acute flares, andimproved quality of life.

In one aspect, the subject invention provides a therapeutic compositionfor treating human dermatological disorders, comprising a bacterialstrain, or a bioactive extract therefrom, and a pharmaceuticallyacceptable excipient. The composition preferably has one or morebiological activities selected from inhibiting anti-microbial activity,pathogenic biofilm growth, inhibiting pathogenic biofilm adhesion,promoting pathogenic biofilm detachment, promoting commensal biofilmgrowth, enhancing skin barrier functions, and enhancing skin innateimmune functions.

In exemplary embodiments, the pathogenic bacterium ismethicillin-resistant Staphylococcus aureus (MRSA) and the commensalbacterium is Staphylococcus epidermidis (S. epidermidis).

In some embodiments, the bacterial strain is Lactobacillus fermentumQi6, also referred to herein as Lf Qi6. In one embodiment, the subjectinvention provides an isolated or a biologically pure culture of Lf Qi6.In another embodiment, the subject invention provides a biologicallypure culture of Lf Qi6, grown as a biofilm. Specifically taught hereinare methods for inducing and identifying the biofilm phenotype. Furtherprovided herein are methods of utilizing the biofilm phenotype, as wellas extracts of the biofilm phenotype, and lysates thereof. In preferredembodiments, the pharmaceutical compositions comprise bioactive extractsof Lf Qi6 biofilm.

In a further aspect, the subject invention provides a cosmeticcomposition for improving human skin conditions, comprising Lf Qi6,and/or a bioactive extract thereof, and together with one or morecosmetically acceptable excipients. In preferred embodiments, thecosmetic composition comprises bioactive extracts of Lf Qi6 biofilm.

In some embodiments, the cosmetically acceptable excipients comprisesubstances used for formulations selected from lotion, cream, emulsion,ointment, oil, gel, serum, and combinations thereof.

In yet another aspect, the subject invention provides a method ofimproving human skin conditions, comprising administering to a subjectan effective amount of a composition comprising a bacterial strain,and/or a bioactive extract thereof, and one or more cosmeticallyacceptable excipients. As with the therapeutic composition, the cosmeticcomposition preferably has one or more biological activities selectedfrom inhibiting pathogenic biofilm growth, inhibiting pathogenic biofilmadhesion, promoting pathogenic biofilm detachment, promoting commensalbiofilm growth, enhancing skin barrier functions, enhancing skin innateimmune functions, and combinations thereof. In preferred embodiments,the bacterial strain is Lf Qi6.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a Lf Qi6 Culture procedure. Step 1: Lf Qi6 cultured in MRSagar plate; Step 2: Lf Qi6 cultured in 5 ml MRS broth for 24 hour at 37°C.; Step 3: 0.1 ml of the culture is transferred into a T-150 tissueculture plate with 25 ml MRS broth; Step 4: 25 mil MRS media is changedevery 48 hours, biofilm Lf Qi6 is grown on the bottom as lawn; Step 5:the culture is grown for 7 days to get a thick biofilm layer on bottom;Step 6: Biofilm layer is scraped out and suspended in fresh medium.Freezer stocks can be made with glycerol and stored in −80°.

FIG. 2 illustrates a Lf Qi6 Scale-up Culture Procedure. Step 1: Biofilmphenotype Lf Qi6 is cultured in 10 ml fresh MRS medium for 24 hour at37°; Step 2: Inoculate 10 ml culture into 25 L MRS medium with 500 gsterile glass wool; Step 3: Culture it for 72 hours in static conditionsat 37° C. Mix the culture every 24 hours with a gentle shaking; Step 4:Harvest the medium and glass wool. Sonicate the glass wool to detachbiofilm cells; Step 5: Centrifuge the cells to concentrate the biofilmLf Qi6. Suspend in sterile water.

FIG. 3 illustrates an example of Lf Qi6 downstream processing. Step 1:50 g biofilm phenotype Lf Qi6 is suspended in 1 L of sterile water; Step2: The suspension is gently mixed for 24 hours at room temperature forpassive release of bioactive substances; Step 3: the mixture is thensonicated for 30 minutes (50 KHz, 200 watt) into a uniform lysate usingan OmniSonic Ruptor 400; Step 4: the sonicated lysate is frozen; Step 5:the frozen lysate is lyophilized into a fine powder.

FIG. 4 illustrates biofilm growth of Lf Qi6 in substrates in scale-upculture.

FIG. 5 illustrates SDS bands showing unique proteins in bioextractprepared from Lf Qi6 biofilm phenotype. Arrows show unique proteins inbiofilm phenotype compared to planktonic phenotype.

FIG. 6 illustrates size-exclusion HPLC showing unique proteins inbioextract prepared from Lf Qi6 biofilm phenotype.

FIG. 7 illustrates size-exclusion HPLC showing unique proteins inbioextract prepared from Lf Qi6 biofilm phenotype.

FIG. 8 illustrates Lf Qi6 biofilm extracts having high anti-biofilmactivity against MRSA.

FIG. 9 illustrates MRSA colony forming units in biofilm.

FIG. 10 illustrates commensal S. epidermidis biofilm promotion by >50kDA Qi6 media fraction (CV staining).

FIG. 11 illustrates S. epidermidis colony forming units in biofilm.

FIG. 12 illustrates commensal S. epidermidis growth control.

FIG. 13 illustrates that Lf Qi507 rapidly detaches MRSA biofilm in an exvivo human skin organotypic culture system.

FIG. 14A illustrates the modulation of pathogenic MRSA vs. commensal S.epidermidis by Lf Qi601 biofilm in a co-culture model in a 6-well platein vitro. FIG. 14B illustrates the modulation of pathogenic MRSA vs.commensal S. epidermidis by Lf Qi601 biofilm in a co-culture model inex-vivo skin explant. Data is represented as the mean±SEM (standarderror of means) and one-way ANOVA was used to determine differencesamong treatment means (P<0.05). Means with the same letter are notsignificantly different from each other (P<0.05, Newman-Keuls multiplecomparison test).

FIGS. 15A-15F illustrate the expression of skin barrier proteinfilaggrin (FLG) in keratinocytes upregulated by bioextracts from Lf Qi6.FIG. 15A shows the isotype control without treatment of the Lf Qi6bioextract. FIG. 15B shows the PBS-base line. FIG. 15C is an ex-vivoskin explant treated with 0.5 mg/mL of Lf Qi6 bioextract. FIG. 15D is anex-vivo skin explant treated with 1 mg/mL of Lf Qi6 bioextract. FIG. 15Eshows the quantitative immunoratio of skin explants treatment with PBS,0.5 mg/mL of Lf Qi6 bioextract, or 1 mg/mL Lf Qi6 bioextract. FIG. 15Fshows the quantitative FLG ELISA of skin explants treatment with PBS,0.5 mg/mL of Lf Qi6 bioextract, or 1 mg/mL Lf Qi6 bioextract. Data isrepresented as the mean±SEM (standard error of means) and one-way ANOVAwas used to determine differences among treatment means (P<0.05). **denotes P<0.01 and *** denotes P<0.001.

FIG. 16 illustrates that Lf Qi6 bioactives increase PPAR beta, and gammain keratinocytes.

FIG. 17 illustrates that LFQi507 increases expression of human betadefensins.

FIG. 18A shows the percent MRSA biofilm inhibition by Lf Qi6 bioextractscompared to vancomycin and meropenem.

FIG. 18B shows the percent reduction in MRSA biofilm adhesion by Lf Qi6bioextracts compared to vancomycin and meropenem.

FIG. 18C shows the percent MRSA biofilm detachment following thetreatment of vancomycin, meropenem, or Lf Qi6 bioextracts.

FIG. 18D shows the inhibition of MRSA biofilm treated with Lf Qi6bioextract in a 6-well plate with coomassie biofilm staining

FIG. 18E shows the MRSA biofilm formed on ex-vivo skin stained withhematoxylin-eosin, the arrow indicating robust biofilm formation. Datais represented as the mean±SEM (standard error of means) and one-wayANOVA was used to determine differences among treatment means (P<0.05).When significant differences were found, means were compared usingNewman-Keuls multiple comparison test. Means with the same letter arenot significantly different from each other.

FIG. 19A represents the upregulation of human beta-defension 2 by the LfQi6 bioextracts.

FIG. 19B shows the upregulation of human beta-defension 3 in ex-vivoskin model treated with Lf Qi6 bioextracts.

FIG. 19C shows the identification of beta-defensin 2 via HPLC fromtissue culture media treated with Lf Qi6 bioextracts. Data isrepresented as the mean±SEM (standard error of means) and one-way ANOVAwas used to determine differences among treatment means (P<0.05). ***denotes P<0.001.

DETAILED DESCRIPTION OF THE INVENTION

The subject invention provides compositions and methods for treatinghuman dermatological conditions. Preferred embodiments of the inventionprovide pharmaceutical and cosmetic compositions, and methods of usingthe same, comprising a strain of Lactobacillus fermentum bacterium,and/or one or more bioactive extracts thereof, derived from humanmicrobiota and capable of growing in biofilm phenotype. The subjectinvention also provides compositions comprising L. fermentum bacterium,and/or bioactive extracts thereof, in a lyophilized, freeze dried,and/or lysate form.

Advantageously, preferred compositions and treatment methods providedherein are effective in treating human skin barrier dysfunctions,dysbiosis, skin innate immune dysfunctions, inflammatory skin diseases,as well as improving the appearance and/or texture of the skin. Subjectsthus treated with this microbiome-centered approach can experiencerelieved symptom severity, reduced incidence of acute flares, andimproved quality of life.

In some embodiments, the bacterial strain is Lactobacillus fermentumQi6, also referred to herein as Lf Qi6. In one embodiment, the subjectinvention provides an isolated or a biologically pure culture of Lf Qi6.In another embodiment, the subject invention provides a biologicallypure culture of Lf Qi6, grown as a biofilm. Specifically taught hereinare methods for inducing and identifying the biofilm phenotype. Furtherprovided herein are methods of utilizing the biofilm phenotype, as wellas extracts of the biofilm phenotype, including lysates thereof. Inpreferred embodiments, the pharmaceutical compositions comprisebioactive extracts of Lf Qi6 biofilm.

A culture of the L. fermentum microbe has been deposited with theAmerican Type Culture Collection (ATCC), 10801 University Blvd.,Manassas, Va. 20110-2209 USA. The deposit has been assigned accessionnumber ATCC No. PTA-122195 by the repository and was deposited on Jun.10, 2015.

The subject culture has been deposited under conditions that assure thataccess to the culture will be available during the pendency of thispatent application to one determined by the Commissioner of Patents andTrademarks to be entitled thereto under 37 CFR 1.14 and 35 U.S.C 122.The deposit is available as required by foreign patent laws in countrieswherein counterparts of the subject application, or its progeny, arefiled. However, it should be understood that the availability of adeposit does not constitute a license to practice the subject inventionin derogation of patent rights granted by governmental action.

Further, the subject culture deposit will be stored and made availableto the public in accord with the provisions of the Budapest Treaty forthe Deposit of Microorganisms, i.e., it will be stored with all the carenecessary to keep it viable and uncontaminated for a period of at leastfive years after the most recent request for the furnishing of a sampleof the deposit, and in any case, for a period of at least 30 (thirty)years after the date of deposit or for the enforceable life of anypatent which may issue disclosing the culture. The depositoracknowledges the duty to replace the deposit should the depository beunable to furnish a sample when requested, due to the condition of thedeposit. All restrictions on the availability to the public of thesubject culture deposit will be irrevocably removed upon the granting ofa patent disclosing it.

As used herein, reference to an “isolated” microbe refers to one thathas been removed from materials with which it exists in nature. Themicrobe may be isolated from, for example, soil, blood, mucous, or milksuch that it is removed from, and is no longer mixed or otherwiseassociated with, those materials to the extent that it is in nature.Such isolation can be used to impart upon the microbe markedly differentcharacteristics, such as the production of different, or differentamounts of, compounds, compared to what the microbe exhibits in itsnatural state.

In one aspect, the subject invention provides a pharmaceuticalcomposition for treating human dermatological disorders, comprising abiologically pure bacterial strain, and/or a bioactive extract thereof,and one or more pharmaceutically acceptable excipients, the bacterialstrain and its extract being capable of growing in both planktonic andbiofilm phenotypes, the composition having one or more biologicalactivities selected from, general antimicrobial activity, inhibitingpathogenic biofilm growth, inhibiting pathogenic biofilm adhesion,promoting pathogenic biofilm detachment, promoting commensal biofilmgrowth, enhancing skin barrier functions, and enhancing skin innateimmune functions.

As used herein the term “extract” refers to a composition obtained byprocessing a biofilm culture. The processing may involve, for example,physical and/or chemical treatment. The physical and/or chemicaltreatment may comprise, for example, filtering, centrifugation,sonication, pressure treatment, radiation treatment, lysing, treatmentwith solvents or other chemicals, and combinations of these treatments.The extract can be in the form of, for example, a supernatant such asthat produced via centrifugation. The extract can also include cell massobtained through centrifugation. The cells may be intact or not intact,viable or not viable. The extract may comprise cell membrane componentsand/or intracellular components. In certain embodiments, the extract isat least 80, 85, 90, or 95%, by weight, cell mass. In certainembodiments, at least 95% of the intact cells are non-viable. In certainembodiments, less than 10% of the cell mass in the extract is intactcells.

Human skin comprises two compartments, the deep compartment (the dermis)and the surface compartment (the epidermis). The skin constitutes abarrier against external attacks, particularly chemical, mechanical, orinfectious attacks, as well as a number of defensive reactions againstenvironmental factors such as, for example, climate, ultraviolet rays,and tobacco, and/or xenobiotic factors, such as, for example,microorganisms. This property is referred to as the skin barrierfunction and is mainly provided by the most superficial layer of theepidermis, namely the horny layer, referred to as the stratum corneum.Detrimental changes in the barrier can be reflected by, for example,cutaneous discomfort, sensory phenomena and/or cutaneous dryness.

Compositions according to some embodiments of the invention are usefulfor preventing a reduction in the barrier function and/or to repair orregenerate barrier function. Disorders associated with disruption of theskin and/or mucosal barrier include, but are not limited to, psoriasis,icthyosis, sarcoidosis, atherosclerosis, inflammatory bowel disease,acne (including hiradenitis suppurativa), burns, diaper rash,Netherton's syndrome, actinic keratosis, dermatomycoses, dermatosis orectodermal dysplasia, atopic dermatitis, contact dermatitis, seborrehicdermatitis, vulgaris, eosinophilic esophagitis, filaggrin deficiency,and other disorders associated with damage or breakdown of the skinand/or mucosal barrier.

In some embodiments, repair or regeneration of the barrier includesrepair or regeneration of a mucous membrane. Mucous membranes includemucosa of the mouth (including mucosa of the cheek, the soft palate, thetongue, including the under surface of the tongue and the floor of themouth), the nose, the throat (including mucosa of the pharynx, thelarynx, the trachea and the esophagus), the bronchi, the lungs, the eye,the ear, the gastrointestinal tract, the vagina, the penis, the urethra,the bladder, and the anus. In certain embodiments, the compositions ofthe subject invention can also be used in the treatment of acute andchronic viral infections. In particular, the treatment of chronicEpstein-Barr virus, cytomegalovirus and other herpes-type virusinfection, which are ubiquitous in the population and are associatedwith a decrease on the immune survillance.

The pharmaceutical compositions provided herein may also include otherpharmaceutically-acceptable ingredients known to those skilled in theart, including, but not limited to, pharmaceutically-acceptablecarriers, adjuvants, excipients, diluents, fillers, buffers,preservatives, anti-oxidants, lubricants, stabilisers, solubilisers,surfactants (e.g., wetting agents), masking agents, and colouringagents. The formulations may further comprise other active agentsincluding, for example, other therapeutic or prophylactic agents.

As provided herein, “pharmaceutically acceptable” refers to approved orapprovable by a regulatory agency of the Federal or a state governmentor listed in the U.S. Pharmacopeia or other generally recognizedpharmacopeia for use in animals, including humans.

“Pharmaceutically acceptable excipient, carrier or adjuvant” refers toan excipient, carrier or adjuvant that can be administered to a subject,together with an active ingredient, and which does not destroy thepharmacological activity thereof and is nontoxic when administered indoses sufficient to deliver a therapeutic amount of the compositionsprovided herein.

In some embodiments, the biologically pure strain is Lactobacillusfermentum Qi6, hereafter also referred to as Lf Qi6. In preferredembodiments, the pharmaceutical compositions provided herein compriseone or more bioactive extracts of Lf Qi6 obtained after it has beengrown in the biofilm phenotype.

“Planktonic” refers to a phenotype typical to microorganisms (bacteria,fungi, and/or protozoa, with associated bacteriophages and otherviruses) that float freely in a liquid medium. A “biofilm,” on the otherhand, is an accumulation of microorganisms embedded in an extracellularpolymeric matrix (EPS) and adherent to solid biological or non-bioticsurfaces. It has been observed and reported in the examples providedherein that Lf Qi6 can form biofilm in addition to possessing thetypical planktonic phenotype.

Methods for growing biofilm are known in the art and are described in,for example, WO 2012/118535, which is incorporated herein, in itsentirety, by reference, including the publications cited in thatreference, such as those cited at pages 26-31.

Whereas biofilms of pathogenic bacteria such as Staphylococcus aureus(S. aureus) can cause highly resistant bacterial infections, biofilms ofLf Qi6 and extracts thereof have been found to have anti-dysbiotic andanti-biofilm (e.g., of pathogenic bacteria) properties, skin barrierenhancement, and immunomodulatory effects on human keratinocytes andex-vivo human skin cultures.

Furthermore, co-culture studies have shown that 0.5-1.0 mg/ml of Lf Qi6biofilm extracts reduce the burden of pathogenic biofilm on the skinwhile promoting commensal bacteria growth. Enhancing the relativeproportion of resident commensal bacteria can decrease the load ofpathogenic biofilm on surfaces.

In exemplary embodiments, the pathogenic bacterium ismethicillin-resistant Staphylococcus aureus (MRSA) and the commensalbacterium is Staphylococcus epidermidis (S. epidermidis).

Infections occur where disease causing microorganisms invade the tissuesof the body. Those microorganisms and the toxins that they produce reactwith the tissues of the body, often causing immune reactions by theinfected host. Infections may be caused by bacteria, viruses, viroids,fungi and other parasites. Infections may occur via any of the tissuesof the body, such as the skin, gut or membranes. In specificembodiments, the subject invention provides compositions for thetreatment and/or prevention of infection of the external surface of thebody, and particularly of the skin. The infection may be caused by abacterium, such as pathogenic Staphylococcus bacteria. Thepharmaceutical compositions provided herein may be applied separately,sequentially or simultaneously with exposure to the infective agent,such as MRSA. In other embodiments, the subject invention providesmaterials and methods for treating intestinal and other internaldisorders.

S. aureus is a transient colonizer of skin predominantly in the moist,warm regions of the body such as the groin, axilla and the anteriornares. Up to 60% of the population are intermittent carriers whileanother 20% may be stably colonized. While normal carriage isasymptomatic, S. aureus may invade tissues (e.g., through broken skin)where it causes diseases ranging from the relatively minor impetigo andscalded skin syndrome, to life threatening conditions such assepticaemia. Furthermore, S. aureus infection is often a secondaryphenomenon in skin with underlying conditions such as atopic dermatitis(AD).

Exemplary compositions provided by the subject invention are useful forthe treatment of infections by a number of pathogenic bacteriaincluding, but not limited to, Staphylococcus spp., Pseudomonas spp.,Staphyloccus saprophyticus, Staphyloccocus xylosus, Staphyloccocuslugdunensis, Staphyloccocus schleiferi, Stapylococcus caprae,Staphylococcus saprophyticus, Staphylococcus hominis, Staphylococcusaureus, Enterococcus faecalis, vancomycin-resistant Enterococcus (VRE),Bacillus cereus, Bacillus subtilis, Listeria monocytogenes,Streptococcus pyrogenes, Streptococcus salivariu, Streptococcus mutans,and Streptococcus pneumonia. Other pathogenic bacteria will be readilyrecognized by a person skilled in the art.

Certain compositions provided herein exhibit anti-S. aureus, preferablyMRSA, activity, and are thus useful for the treatment or prevention ofS. aureus infection, including the inhibition of bacterial growth,inhibition of bacterial adhesion, and promotion of bacterial detachment.

In accordance with the subject invention, Lf Qi6 has been found to haveanti-adhesive, inhibition and detachment activities against MRSAbiofilm, both in vitro and in a living ex-vivo human skin explant model.There are several potential mechanisms for Lf Qi6 MRSA anti-biofilmactivity. For instance, Lf Qi6, or its extracts, may containanti-biofilm peptides or heat shock proteins, and/or (p)ppGpp-blockers.Proteomic analysis from a whole genome sequencing of Lf Qi6 suggestsseveral unique heat shock proteins and other related stress proteins.

Some embodiments of the subject invention provide that the enhancementof skin barrier function is achieved by upregulating the expression ofskin barrier proteins. In other embodiments, the enhancement of skininnate immune functions is provided by modulating the expression of skininnate immune peptides and/or inflammatory cytokines.

In some embodiments, bioactives extracted from Lf Qi6 biofilm haveagonistic activities towards peroxisome proliferator-activated receptors(PPARs), namely, PPAR-alpha, PPAR-beta/delta, and/or PPAR-gamma. PPARsare a group of nuclear receptor proteins and serve as transcriptionfactors to regulate gene expression in response to various physiologicalstimuli. Their structure is highly conserved, composed of anamino-terminal activation domain (AF1), a zinc-finger DNA bindingdomain, a ligand-binding carboxy-terminal domain and a second activationdomain at the c-terminus (AF2).

PPAR-alpha is expressed in metabolically active tissues such as brownfat, liver, heart, muscle, kidney, immune cells. Its ligands includedocosahexanoic acid (DHA), WY 14643, clofibrate, oxidized phospholipids,phthalate esters, and various herbicides. The first three ligands areknown to increase filaggrin and heat shock protein 27 (HSP27).PPAR-alpha is a major regulator of hepatic lipid metabolism, activatedunder conditions of energy deprivation to initiate ketogenesis, anadaptation to prolonged fasting. PPAR-alpha synthetic ligands includethe anti-hyperlipidemic fibrate drugs. Because PPAR-alpha ligandsregulate hepatic lipid metabolism, they may have utility in thetreatment of steatohepatisis, or fatty liver.

PPAR-beta/delta is expressed in metabolically active tissues includingskin, intestinal tract, liver, heart, skeletal muscle, lung, brain,thymus, spleen, keratinocytes and various immune cells. Its ligandsincludes GW 1514 and retinoic acid. Agonism of PPAR-beta/delta changesthe body's fuel preference from glucose to lipids, but also has beendemonstrated to play a role in myelination of the corpus callosum,epidermal cell proliferation, as well as differentiation, lipidaccumulation, directional sensing, polarization, and migration inkeratinocytes.

PPAR-gamma is ubiquitously expressed, though mainly present in adiposetissue, colon and macrophages. Because its ligands include thethiazolidinedione (TZD) rosiglitazone, pioglitazone, troglitazone, PPARgamma is also referred to as the “glitazone” receptor. As a regulator ofadipocyte differentiation, it is important as a regulator of fatty acidstorage and glucose metabolism. Compounds with activities at thePPAR-gamma site have particularly valuable pharmacologic potential asoral, injectable or otherwise systemic treatments for diabetes, insulinresistance, metabolic syndrome, obesity, atherosclerotic heart diseaseand other dysmetabolic states. Ligand-mediated activation of the PPARgamma receptor is although though to be responsible for inhibiting thegrowth of cultured human breast, gastric, lung, prostate and othercancer cell lines (46)

Because PPARs have significant impact on anti-apoptosis and celldifferentiation, anti-inflammatory activity, and lipid and glucosemetabolism, embodiments of the subject invention also provide that LfQi6 bioextracts have particularly valuable utility as a local orsystemic pharmacologic agent in a variety of disorders benefiting fromPPAR agonism, including dysmetabolic, chronic inflammatory states,obesity, insulin resistance, diabetes, metabolic syndrome,atherosclerosis, steatohepatitis, Alzheimer's disease, as well as skindisorders, such as hair loss, acute and chronic wounds, diseases ofchronic skin inflammation such as atopic dermatitis, rosacea, acne,sebhorrheic dermatitis, and diseases thought to involve airwayneutrophilia, such as acute respiratory distress syndrome (ARDS) andchronic obstructive pulmonary disease (COPD). Treatment of metabolicdisorders can include, for example, modulation of lipid, glucose and/orfatty acid storage.

In some embodiments of the current invention, the enhancement of skinbarrier functions is achieved by upregulating the expression of one ormore skin barrier proteins selected from, for example, filaggrin 1,filaggrin 2, loricrin, involucrin, junction proteins, and/or desmosomalproteins.

In some embodiments, the enhancement of skin innate immune functions isachieved by modulating the expression of one or more innate immunepeptides and/or inflammatory cytokines, which have been associated withreduced pathogenic biofilm burden on skin surfaces.

In some embodiments, the innate immune peptides are selected from humanbeta defensin 1, human beta defensin 2, human beta defensin 3, humancathelicidins (LL-37), and combinations thereof.

In some embodiments, the inflammatory cytokines are selected frominterleukin-1 alpha, interleukin-4, interleukin-13, thymic stromallymphoproteins (TSLP), and combinations thereof.

Other proteins, peptides, and cytokines modulating the human skinbarrier, immune, and anti-inflammatory functions will be readilyrecognized by the person skilled in the art.

In certain embodiments, the subject invention also providesantibacterial compositions in the form of cleaning products, washes,surface coatings or other compositions, which are not for medicaltreatment of the human or animal body. Thus, in specific embodiments,these compositions are used to disinfect inanimate surfaces.

In another aspect, the subject invention provides a method of treatinghuman dermatological disorders comprising administering to a subject atherapeutically effective amount of the pharmaceutical composition, thecomposition preferably comprising one or more bioactive extracts of theLf Qi6 biofilm.

“Subject”, as used herein, means a human or a non-human animal, e.g.,dogs, cats, mice, rats, cows, sheep, pigs, goats, non-human primates orbirds, e.g., a chicken, as well as any other vertebrate or invertebrate.

Administration of the pharmaceutical compositions provided herein ispreferably in a “therapeutically effective amount,” this being an amountsufficient to result in a biological or medical response of a cell,tissue, system, animal, or human that is being treated. The actualamount administered, and the rate and time-course of administration,will depend on the nature and severity of the disease being treated aswell as the subject. Prescription of treatment, e.g., decisions ondosage etc, is within the responsibility of general practitioners andother medical doctors, and typically takes into account the disorder tobe treated, the condition of the individual patient, the site ofdelivery, the method of administration and other factors known topractitioners.

A composition may be administered alone or in combination with othertreatments, either simultaneously or sequentially dependent upon thecondition to be treated. The pharmaceutical compositions provided hereinmay be dissolved in, suspended in, or admixed with one or more otherpharmaceutically acceptable ingredients. The compositions may also bepresented in a liposome or other microparticle.

In preferred embodiments, the pharmaceutical compositions are formulatedfor topical administration, particularly for use or application to, oron, the skin. Such formulations may be useful for removing, killing, orpreventing the adhesion and accumulation of pathogenic bacteria, such asMRSA, on a biological or non-biotic surface, or inhibiting the action orgrowth of the bacteria. Furthermore, in specific embodiments,compositions comprising biofilm, or the extracts thereof, of Lf Qi6 havethe advantage of promoting the growth of commensal bacteria in the humanskin microbiome. Non-limiting examples of the commensal bacteriainclude, but are not limited to, Staphylococcus epidermidis (S.epidermidis), Staphylococcus warneri, Streptococcus mitis,Propionibacterium acnes, Corynebacterium spp., Acinetobacter johnsonii,and Pseudomonas aeruginosa.

The pharmaceutical compositions provided herein may suitably be providedas a patch, adhesive plaster, bandage, dressing, or the like which isimpregnated with, or coated with, the microbial biofilm and/or one ormore extracts thereof and, optionally, one or more otherpharmaceutically-acceptable ingredients, including, for example,penetration, permeation, and absorption enhancers.

Antibacterial compositions according to embodiments of the invention maybe useful for treating biomaterials, implants and prosthesis (includingstents, valves, eyes, hearing aids, gastric bands, dentures, artificialjoint replacements etc.), surgical instruments or other medical devicesprior to administration to, or treatment of, or use with, a subject. Theantibacterial compositions may be useful for treating surfaces prone tocolonization or exposure to bacteria, such as handrails, foodpreparation surfaces, kitchen surfaces or equipment, tables, sinks,toilets or other bathroom hardware.

Antibacterial compositions may comprise agents in addition to themicrobial (e.g., Lf Qi6) biofilm or its bioactive extracts, such ascleaning agents, stabilisers, anionic surfactants, perfumes, chelatingagents, acids, alkalis, buffers or detergents. Such agents mayfacilitate or enhance the antibacterial properties of the compositions,such as killing or inhibiting bacteria, or preventing the recolonizationof the cleaned surface.

Exemplary embodiments provide that the subject is one that is affectedby conditions selected from skin barrier dysfunctions, skin dysbiosis,skin innate immune dysfunctions, and combinations thereof.

Embodiments provided herein are useful in the treatment of, for examplepsoriasis, icthyosis, sarcoidosis, atherosclerosis, inflammatory boweldisease, acne (including hiradenitis suppurativa), dermatitis, woundhealing, acne (including hiradenitis suppurativa), burns, diaper rash,Netherton's syndrome, actinic keratosis, dermatomycoses, dermatosis orectodermal dysplasia, atopic dermatitis, contact dermatitis, sebortheicdermatitis, vulgaris, eosinophilic esophagitis, filaggrin deficiency,and/or other disorders associated with damage or breakdown of the skinbarrier.

In some embodiments, repair or regeneration of the barrier includesrepair or regeneration of a mucous membrane. Mucous membranes includemucosa of the mouth (including mucosa of the cheek, the soft palate, thetongue, including the under surface of the tongue and the floor of themouth), the nose, the throat (including mucosa of the pharynx, thelarynx, the trachea and the esophagus), the bronchi, the lungs, the eye,the ear, the gastrointestinal tract, the vagina, the penis, the urethra,the bladder, and the anus.

In certain embodiments, the compositions of the subject invention canalso be used effectively in the treatment of acute and chronic viralinfections. In particular, the treatment of chronic Epstein-Barr virus,cytomegalovirus and other herpes-type virus infection, which areubiquitous in the population and are associated with a decrease on theimmune survillance.

In a further aspect, the subject invention provides a cosmeticcomposition for improving human skin conditions, comprising abiologically pure strain of a bacterium and/or one or more bioactiveextracts thereof, and cosmetically acceptable excipients. In preferredembodiments, the composition comprises bioactive extracts of Lf Qi6biofilm.

“Cosmetic” as used herein is non-therapeutic. Such compositions are notdirected to treating human or animal by therapy. For example, thecomposition may enhance the hydration level of the skin. Increasedhydration level is known to improve the appearance of the skin and leadto a healthier cosmetic appearance.

In some embodiments, the cosmetically acceptable excipients comprisesubstances used for formulations selected from lotion, cream, emulsion,ointment, gel, serum, and combinations thereof.

Cosmetic compositions according to embodiments of the subject inventionhave barrier maintenance and repair activity. As such, they are usefulfor preventing a reduction in and/or reinforcing barrier function. Thismay be useful for improving hydration of the skin and/or improving theappearance of the skin.

Formulations suitable for dermal and/or transdermal administrationinclude, but are not limited to, gels, pastes, ointments, creams,lotions, and oils, as well as patches, adhesive plasters, bandages,dressings, depots, cements, glues, and reservoirs.

Ointments are typically prepared from the cosmetic compositions providedherein and a paraffinic or a water-miscible ointment base.

Creams are typically prepared from the cosmetic compositions providedherein and an oil-in-water cream base. If desired, the aqueous phase ofthe cream base may include, for example, at least about 30% w/w of apolyhydric alcohol, i.e., an alcohol having two or more hydroxyl groupssuch as propylene glycol, butane-1,3-diol, mannitol, sorbitol, glyceroland polyethylene glycol and mixtures thereof.

As would be readily appreciated by one skilled in the art, formulationsaccording to the subject invention could also comprise other alcohols,such as, for example, isopropyl alcohol or ethanol, and could also coverother alcohol based formulations, for example alcohol based handsanitizers.

The topical formulations may desirably include a compound that enhancesabsorption or penetration of the active compound through the skin orother affected areas. Examples of such dermal penetration enhancersinclude dimethylsulfoxide and related analogues.

Emulsions are typically prepared from the cosmetic compositions providedherein and an oily phase, which may optionally comprise merely anemulsifier (otherwise known as an emulgent), or it may comprise amixture of at least one emulsifier with a fat or an oil or with both afat and an oil. Preferably, a hydrophilic emulsifier is includedtogether with a lipophilic emulsifier which acts as a stabiliser. It isalso preferred to include both an oil and a fat. Together, theemulsifier(s) with or without stabiliser(s) make up the so-calledemulsifying wax, and the wax together with the oil and/or fat make upthe so-called emulsifying ointment base which forms the oily dispersedphase of the cream formulations.

Suitable emulgents and emulsion stabilisers include Tween 60, Span 80,cetostearyl alcohol, myristyl alcohol, glyceryl monostearate and sodiumlauryl sulphate. The choice of suitable oils or fats for the formulationis based on achieving the desired cosmetic properties, since thesolubility of the active compound in most oils likely to be used inpharmaceutical emulsion formulations may be very low. Thus the creamshould preferably be a non-greasy, non-staining and washable productwith suitable consistency to avoid leakage from tubes or othercontainers. Straight or branched chain, mono- or dibasic alkyl esterssuch as di-isoadipate, isocetyl stearate, propylene glycol diester ofcoconut fatty acids, isopropyl myristate, decyl oleate, isopropylpalmitate, butyl stearate, 2-ethylhexyl palmitate or a blend of branchedchain esters known as Crodamol CAP may be used, the last three beingpreferred esters. These may be used alone or in combination depending onthe properties required. Alternatively, high melting point lipids suchas white soft paraffin and/or liquid paraffin or other mineral oils canbe used. Other formulations include dental sprays, mouthwashes,toothpastes, lozenges, antibacterial washes, drinks (e.g., milk,yoghurt), food items (such as yoghurt, ice cream, candy bars), orpowdered foods (such as powdered milk).

In some embodiments, the cosmetic composition has biological activitiesselected from inhibiting pathogenic biofilm growth, inhibitingpathogenic biofilm adhesion, promoting pathogenic biofilm detachment,promoting commensal biofilm growth, enhancing skin barrier functions,enhancing skin innate immune functions, and combinations thereof.

In yet another aspect, the subject invention provides a method ofimproving human skin conditions, comprising administering to a subject acosmetically effective amount of a composition comprising a biologicallypure bacterial strain, and/or one or more bioactive extracts thereof,and cosmetically acceptable excipients, the bacterial strain and itsextract being capable of growing in both planktonic and biofilmphenotypes, the composition having biological activities selected frominhibiting pathogenic biofilm growth, inhibiting pathogenic biofilmadhesion, promoting pathogenic biofilm detachment, promoting commensalbiofilm growth, enhancing skin barrier functions, enhancing skin innateimmune functions, and combinations thereof. In preferred embodiments,the bacterial strain is Lf Qi6.

The cosmetic treatment may be used to improve the appearance and/ortexture of the skin. In an exemplary embodiment, the method relates toimproving the hydration level, or appearance, of the skin. As usedherein the term “cosmetic method” does not refer to a method fortreatment of the human or animal body by surgery or therapy, ordiagnostic methods practiced on the human or animal body.

The subject to be treated may be any animal or human. The subject may bea non-human mammal, but is more preferably a human. The subject may bemale or female. In some embodiments, the subject does not require repairof his or her skin barrier, or to be treated for infection, such asbacterial infection. In some embodiments, the subject does not requiretreatment at the site at which the cosmetic treatment is to be applied.

The cosmetic methods according to the invention preferably involve theadministration of a “cosmetically effective amount”. This pertains tothe administration of compounds, ingredients, materials, compositions,dosage forms, etc. in an amount effective to induce a cosmetic benefit.This is within the scope of sound judgement of a relevant practitioner.It will be appreciated by one of skill in the art that appropriatedosages of the active compounds, and compositions comprising the activecompounds, can vary from subject to subject.

The pharmaceutical or cosmetic compositions provided herein may containa single (unit) dose of probiotic bacteria, or lysate, or extractthereof. Suitable doses of probiotic bacteria (intact, lysed orextracted) may be in the range 104 to 1012 cfu, e.g., one of 104 to1010, 104 to 108, 106 to 1012, 106 to 1010, or 106 to 108 cfu. In someembodiments, doses may be administered once or twice daily. In someembodiments, a composition for use according to the present inventionmay comprise at least about 0.01%, about 0.05%, about 0.1%, about 0.2%,about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%,about 0.9%, about 1.0%, about 1.5%, about 2.0%, about 3.0%, about 4.0%,about 5.0%, about 6.0%, about 7.0%, about 8.0%, about 9.0%, about 10.0%, about 1 1.0%, about 12.0%, about 13.0%, about 14.0%, about 15.0%,about 16.0%, about 17.0%, about 18.0%, about 19.0%, about 20.0%, about25.0%, about 30.0%, about 35.0%, about 40.0%, about 45.0%, about 50.0%by weight of the Lf Qi6 extracts. In some embodiments, the compositionsmay comprise, one of at least about 0.01% to about 30%, about 0.01% toabout 20%, about 0.01% to about 5%, about 0.1% to about 30%, about 0.1%to about 20%, about 0.1% to about 15%, about 0.1% to about 10%, about0.1% to about 5%, about 0.2% to about 5%, about 0.3% to about 5%, about0.4% to about 5%, about 0.5% to about 5%, about 1% to 10 about 5%, byweight of the Lf Qi6 extracts.

For the purpose of the present invention the abbreviation cfu shalldesignate a “colony forming unit” that is defined as the number ofbacterial cells as revealed by microbiological counts on agar plates.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety, including all figures and tables, to the extent theyare not inconsistent with the explicit teachings of this specification.

Materials and Methods Bacterial Strains and Culture Media

Methicillin-resistant Staphylococcus aureus (MRSA) ATCC 33591 (ATCC,Manassas, Va.) was stored in tryptone soya broth (TSB) (ThermoScientific, Waltham, Mass.) containing 20% (v/v) glycerol at −80° C.Culture was incubated overnight at 37° C., aerobic, on a rotary shakerat 110 rpm. Optical density of the overnight culture was read withspectrophotometer (SpectraMax Plus384, Molecular Devices, Sunnyvale,Calif.) and diluted to 0.2OD₆₀₀. The strain was also subjected to bothmet and eap PCR using established protocols to confirm MRSA status (23).Lactobacillus fermentum Qi6 (Lf Qi6) was grown in MRS media at 37′C. LfQi6 is a proprietary strain. The genome of this strain is sequenced(GenBank Accession No. LAIK00000000). The descriptions of the Lf Qi6genome sequence are incorporated by reference in their entirety (24),and specifically included therein is the web access to the whole genomesequence from http://www.ncbi.nlm.nih.gov/nuccore/LAIK00000000.Staphylococcus epidermidis K7 was isolated and identified from humanmicrobiota, and the commensal nature of the strain is verified usingstandard PCR tests for mecA, fdh, esp, eap genes (25-26).

Culture of Lf Qi6 Biofilm

FIG. 1 shows procedures for seeding the Lf Qi6 culture. After beingisolated and identified, Lf Qi6 was cultured in MRS agar plate. Theculture was then incubated in 5 ml of MRS broth for 24 hour at 37° C. 1ml of the culture was transferred into a T-150 tissue culture plate with25 ml of MRS broth. 25 ml of MRS media was changed every 48 hours toallow the biofilm of Lf Qi6 to grow as lawn on the bottom of the cultureplate. The culture was then grown for 7 days to produce a thick biofilmlayer. The grown biofilm layer was subsequently scraped out andsuspended in fresh medium. Freezer stacks were made with glycerol andstored in −80°.

A scaled-up production of Lf Qi6 biofilm is illustrated in FIG. 2. Abiofilm phenotype of Lf Qi6 in frozen stock was cultured in 10 ml offresh MRS media for 24 hour at 37°. 10 ml of culture was inoculated into25 L of MRS media with 500 g sterile glass wool. The biofilm was thencultured for 72 hours under static conditions at 37° C. The culture wasmixed every 24 hours with a gentle shaking, after which the media andglass wool were harvested. The biofilm cells were subsequently detachedfrom the glass wool via sonication. The cells were further centrifugedto concentrate the biofilm of Lf Qi6, which was then suspended insterile water. This scale-up yields a biofilm culture at a concentrationof 50 g/25 L. The Lf Qi6 biofilm growth is further illustrated in FIG.4, wherein the biofilm was cultured on substrates in a scaled-up cultureas described herein.

Lf Qi6 Biofilm Downstream Processing

The downstream processing of Lf Qi6 biofilm is shown in FIG. 3. 50 g ofbiofilm phenotype of Lf Qi6 was suspended in 1 L of sterile water. Thesuspension was gently mixed for 24 hours at room temperature to allowthe passive release of multiple bioactives. The mixture was thensonicated for 30 minutes (50 KHz, 200 watt) into uniform lysate using anOmniSonic Ruptor 400. The sonicated lysate was then frozen andlyophilized into a fine powder.

Culture of Human Keratinocyte Cell Line

HEKa cells were purchased from Life Technologies (Grand Island, N.Y.,USA Cat# C-005-5C). HEKa cells were maintained in Medium 154 (LifeTechnologies, Grand Island, N.Y., USA Cat#M-154-500) supplemented withHuman Keratinocyte Growth Supplement (Cat#S-001-5).

Human Ex-Vivo Organotypic Culture

Surgical specimens of human skin were obtained post-cosmetic surgery.All samples were obtained from patients who gave informed consent to useexcess skin specimens for research purposes. Skin was transported to thelaboratory in fresh Eagle's minimum essential medium (EMEM) withL-glutamine (ATCC, Manassas, Va.) supplemented with 1%antibiotic-antimycotic solution (penicillin, streptomycin, amphotericin)(Hyclone, Thermo Scientific, Logan, Utah). The medium and skin werestored at 4° C. prior to cutting). Culture medium was composed ofKeratinocyte-complete serum free medium supplemented with humanrecombinant Epidermal Growth Factor (0.2 ng/ml EGF), Bovine PituitaryExtract (30 lg/ml BPE) (Life technologies, Grand Island, N.Y.), and 1%antibiotic-antimycotic solution. 6-well plates were prepared with 1.3 mlculture medium and one Millicell-CM cell culture insert (0.4 μm, 30 mmdiameter) (EMD Millipore, Billerica, Mass.) per well. Explant sampleswere prepared for cutting by washing gently in fresh supplemented EMEMin a sterile petri dish. Explants were moved with sterile forceps to afresh dish containing EMEM, cut into 1 cm² pieces using sterile surgicalscissors and placed over cell culture inserts in 6-well plates so theunderside portions of the skin were exposed to air (air-liquidinterface). Explants were cultured at air-liquid interface on cellculture inserts at 37° C. with 5% CO₂ for 5-7 days, based onexperimental protocol, with medium changed daily.

Preparation of Lf Qi6 from Problotic Bacteria

L. fermentum Qi6 was grown in MRS media using proprietary culturemethods. Bacteria were then subcultured into 500 ml MRS medium for anadditional period, again using proprietary culture methods. Bacteriawere sonicated (Reliance Sonic 550, STERIS Corporation, Mentor, Ohio,USA), centrifuged at 10,000 g, cell pellets dispersed in sterile water,harvested cells lysed (Sonic Ruptor 400, OMNI International, Kennesaw,Ga., USA) and centrifuged again at 10,000 g, and soluble fractioncentrifuged (50 kDa Amicon Ultra membrane filter, EMD MilliporeCorporation, Darmstadt, Germany, Cat#UFC905008). The resulting fractionwas distributed into 0.5 ml aliquots, flash frozen in liquid nitrogenand stored at −80° C.

Biofilm Inhibition Assay

MRSA was added to the wells of sterile polystyrene, tissue-culture (TC)treated, flat-bottom plates (Genesee Scientific, San Diego, Calif., Cat#25-109). TSB served as sterility control. Growth control wells receivedequal parts MRSA and culture medium. Chosen concentrations of Lf Qi601or other test agents were added, the plate incubated at 37° C. for 18 hand then biofilm quantified as described in the staining and biofilmquantification section.

Biofilm Detachment Assay

MRSA was added to the wells of a sterile TC plate and incubated at 37°C. for 18 hr. The plate was washed three times with IX phosphatebuffered saline (PBS) (Thermo Scientific, Waltham, Mass.) using BioTek405 Select LS Microplate Washer (BioTek, Winooski, Vt.). Fresh TSB wasadded to each well. Chosen concentrations of Lf Qi6 were added, then theplate placed in the incubator at 37° C. for 10 min. Remaining biofilmwas stained and quantified as described below.

Anti-Biofilm Adhesion Assay

Wells of a TC plate were coated with chosen concentrations of Lf Qi6 orother test agents; growth control and negative control wells receivedPBS, and incubated overnight at 4° C. static. Plates were washed threetimes with PBS using BioTek microplate washer. After washing, MRSA wasadded, and the plate incubated at 4° C. for 5 h. Adhered biofilm wasstained and quantified.

Staining and Biofilm Quantification

The plate was washed three times with PBS using the BioTek plate washerand placed in a 47° C. incubator for one hour to heat-fix biofilm. Theplate was cooled to room temperature, stained with 0.1% (v/v) crystalviolet for 15 min, then washed with deionized H₂O using the microplatewasher. 100% ethanol was added for 30 min in order to dissolve crystalviolet stain. The plate was read at 590 nm and 600 nm using aspectrophotometer (SpectraMax Plus384, Molecular Devices, Sunnyvale,Calif.).

Ex Vivo Skin Biofilm Studies

Histological Examinations of biofilms in ex vivo skins were confirmedusing the Hematoxylin-Eosin Staining protocol developed by Hochstim etal. (2010) (27). Ex vivo skin cultures were treated with chosenconcentrations of Lf Qi6, inoculated with 100 μl of either low or highdensity MRSA cultures, and grown overnight. Skin tissues were thenhomogenized, diluted, and cultured on TSB plates to quantify colonyforming units (CFUs) per sample.

In Vitro Pathogenic and Commensal Co-Culture Studies

Six-well plates were treated with Lf Qi6 or bacterial cultures for 5 h.MRSA and S. epidermidis K7 inoculum of identical optical densities (ODs)were prepared in TSB. Two mls of these mixtures were inoculated intriplicate in treated vs control wells of 6-well plates.

Ex Vivo Pathogenic and Commensal Co-Culture Studies on Human SkinExplants

Skin explants surfaces were treated with Lf Qi6, the chemical detergent0.2% Triton as positive control, or PBS as negative control, andincubated overnight at 37° C. at 5% CO₂. MRSA and K7 cultures ofidentical optical densities were prepared in TSB. The skin surface wasinoculated with 50 μl of each bacterial culture and incubated overnightat 37° C. Skin tissues were then homogenized, diluted, and cultured onTSB and ORSAB plates to quantify and differentiate pathogenic vs.commensal CFU's per tissue.

Filaggrin ELISA

A human filaggrin ELISA kit (EIAab, Wuhan, China, Cat#1186h) was used toquantify filaggrin. Treated skin explant tissues were homogenized with 1ml PBS mixed with protease inhibitor cocktail (Thermo Scientific,Product #78425), and frozen overnight at −20° C. Samples were thensubjected to two freeze thaw cycles to break cell membranes. Homogenateswere centrifuged at 10000×g for 5 min. Supernatant was aliquoted andstored at −20° C./−80° C. for 2-5 days. Manufacturer's directions werefollowed to quantify levels of filaggrin in homogenates of the skinexplant. The assay was performed in triplicate/sample condition. ELISAresults were analyzed on Molecular Device's SpectraMax Plus384 usingSoftMax® Pro software (Molecular Devices, Sunnyvale, Calif., USA).Statistical analysis was performed using GraphPad Prism version 6.03(GraphPad Software, Inc., La Jolla, Calif., USA).

Immunohistochemistry

Morphological and immunofluorescent analyses were performed on 5 μmparaffin-embedded NEM sections. For analysis of morphology, skinexplants were fixed in 10% formalin, sectioned, deparaffinized,rehydrated and stained with hematoxylin and eosin (HE). For analysis offilaggrin expression, sections were cut, deparaffinized and rehydrated,followed by heat-mediated antigen retrieval. After blocking non-specificbinding using PBS containing 1% bovine serum albumin (BSA, Sigma) and 2%normal human serum (NHS, Sanquin, Leiden, the Netherlands), the sectionswere incubated overnight at 4° C. with primary antibody for filaggrin(1:1000; Covance, Rotterdam, the Netherlands). After washing with PBS,sections were incubated with secondary goat antibody anti-rabbit(Rhodamine Red, 1:300, Jackson Immuno Research, Amsterdam, theNetherlands). Sections were then incubated with streptavidin peroxidasefor 10 min. at room temperature and rinsed with buffer 4 times. DABchromogen was added to sections to visualize staining for 5 min. Slideswere counterstained with hematoxylin solution for Imin. Negative controlwas prepared without addition of antibody. Isotype controls were alsoincluded in the protocol. The sections were mounted with Vectashieldcontaining DAPI for visualization of the nuclei (Vector Laboratories,Amsterdam, the Netherlands). Quantitative Immunoratio analysis fromvarious 8-9 fields from immunostained slides were done by modifying theprotocol developed by Tuominen et al (2010) (28).

RNA Isolation, cDNA Synthesis and qPCR Analysis

Tissue explants for qRT-PCR were flash frozen in liquid nitrogen. TotalRNA was isolated and purified using RNAqueous-4PCR kit (Ambion)according to manufacturer's instructions and stored at −80° C. untilreverse transcription. For real-time PCR analysis, cDNA was synthesizedfrom 0.1 μg of total RNA using high capacity cDNA Reverse Transcriptionkit (Applied Biosystems, Foster City, Calif.) according tomanufacturer's instructions. Quantitative PCR was performed using PowerSYBR Green PCR master mix on a 7500 Real-Time PCR system (AppliedBiosystems). Primers were chosen from the literature (Table. S1) andobtained from IDT technologies. Cycling conditions were as follows: 2mins at 50° C., 10 mins at 95° C., and 40 cycles of 15 sec at 95° C. and1 min at 60° C. Reaction was followed by a dissociation step and meltingcurves were assessed for primer specificity. PCR for each template wasperformed in triplicate in 96-well plates. The mRNA expression levelswere normalized to β-microglobulin reference gene using the comparativeCT (ddCT) method with Data Assist™ software (Applied Biosystems).Negative control reactions without template were included for eachprimer combination.

Statistical Analysis

Quantitative data of various parameters are presented as the mean±SEM(standard error of means) in figures. Statistically significantdifferences in various parameters were determined by one-way ANOVA test.When significant differences (P<0.05) existed, the means were comparedusing the Newman-Keuls multiple comparison test. Statistical softwareGraphPad Prism (GraphPad Software, Inc., San Diego, Calif.) was used toperform statistical analyses.

Our qRT-PCR results showed approximately 250 fold upregulation infilaggrin in skin treated with bioextract compared to PBS baseline. Weconfirm the increased filaggrin protein production via ELISA andimmunohistochemistry.

Following are examples that illustrate procedures for practicing theinvention. These examples should not be construed as limiting.

Example 1—Lf Qi6 Biofilm Phenotype Differs from the Planktonic Phenotype

Product extraction and protein estimation demonstrate different proteinlevels between the biofilm and planktonic phenotypes, as seen in Table 1below. SDS data in FIG. 5 further corroborate such differences, as shownby extra bands expressed in the Lf Qi6 biofilm phenotype that are notpresent in the planktonic phenotype, indicative of unique proteins inthe bioextract of the biolfilm. Size-exclusion HPLC data in FIGS. 6 and7 illustrate differences in molecular weight between proteins inbioextract prepared from Lf Qi6 biofilm and planktonic phenotype.

TABLE 1 Product extraction and protein estimation from biofilm andplanktonic shows different protein levels. Extract Protein ProteinSample Yield Yield % In No. Description (g/L) (g/L) Extract Pellet 1Planktonic 0.4 0.2 52.2 2 Biofilm-unextracted 0.7 0.4 61.3 3Biofilm-extracted 0.6 0.2 37.3 Cell Lysate 1 Planktonic 0.09 0.03 30.8 3Biofilm-extracted 0.10 0.04 34.7 4 Biofilm-unextracted 0.2 0.06 31.6Biosurfactant 1 Planktonic 0.12 0.05 42.2 3 Biofilm-extracted 0.16 0.0636.7

The anti-biofilm activity against MRSA was evaluated between the Lf Qi6biofilm and planktonic phenotypes in 6-well plates, as shown in FIG. 8,with the negative control being a GC-rich culture of MRSA grown in TSBand the positive control being an MRSA-biofilm treated with vancomycin.The MRSA culture was inoculated with 0.5% glucose and incubatedovernight at 37° C. The Lf Qi6, at 0.5 mg/mL and 1.0 mg/mL,respectively, in both planktonic or biofilm whole cells phenotypes, waspre-coated in each well for 2 hours at 4° C. Included in the observationwas >50 kDa residue of spent Lf Qi6 cultures at two differentconcentrations for each phenotype. It is evident that the extracts ofthe Lf Qi6 biofilm phenotype demonstrated inhibition of MRSA biofilmgrowth at both 0.5 and 1.0 mg/mL. These results were quantified andpresented in FIG. 9, wherein the colony forming units of MRSA weresignificantly reduced by the Lf Qi6 biofilm culture medium as comparedto both the control and the planktonic culture medium.

The promotion of commensal bacteria S. epidermidis by Lf Qi6 mediafraction (>50 kDa of extract) was evaluated for both the planktonic andthe biofilm culture medium (FIG. 10). The result clearly demonstratesthe advantage of the biofilm medium at enhancing the growth of S.epidermidis at greater than 70%, while the planktonic phenotype was onlyless than 40% effective in such promotion of commensal biofilm growth.Quantitative colony forming units for S. epidermidis in the presence ofthe Lf Qi6 biofilm and planktonic culture medium are compared in a chartshown in FIG. 11. Furthermore, FIG. 12 shows the results of a visualcomparison between the control (i.e., S. epidermidis growth culture), S.epidermidis with Lf Qi6 bioextract (>50 kDa) from planktonic culturemedium, and S. epidermidis with Lf Qi6 bioextract (>50 kDa) from biofilmculture medium.

Example 2—Lf Qi6 Biofilm has Anti-Biofilm Activities Against MRSA InVitro: Inhibition, Anti-Adhesion, and Detachment

Various anti-biofilm activities of Lf Qi6 were initially assessed invitro via 96-well plate assays, including inhibition, anti-adhesion anddetachment. The biofilm inhibition assay evaluates the new biofilmforming capacity of MRSA when co-incubated with Lf Qi6. Resultsdemonstrate that Lf Qi6 at 0.5-1.0 mg/mL inhibits 40-50% of MRSA biofilmformation measured at 24-hour co-incubation (FIG. 19A). Theanti-adhesion assay evaluates the ability of Lf Qi6 to prevent MRSA frombinding to a surface. Overnight pretreatment with 0.5-1.0 mg/ml Lf Qi6followed by a 5-hour MRSA biofilm exposure blocked 80-85% MRSA biofilmadhesion in a 24-hour culture assay (FIG. 19B). The biofilm detachmentassay demonstrates the ability of Lf Qi6 to remove a preformed biofilm;at 5 minutes of treatment, 0.5-1.0 mg/mL of Lf Qi6 detached 30% of MRSAbiofilm (FIG. 19C), with similar results obtained in a larger formattissue culture plate (6-well plate, 8.5 cm² FIG. 19D).

Example 3—Lf Qi6 Rapidly Detaches MRSA Biofilm in an Ex Vivo Human SkinOrganotypic Culture System

An ex vivo organotypic human skin culture system was optimized for thepurpose of biofilm inoculation onto living human skin explants obtainedfrom surgical specimens. MRSA biofilm cultures were inoculated onto theex vivo surface and allowed to grow for 48 hours. Biofilm was visualizedvia stain, with robust biofilms forming on the skin in 24 to 48 hours(FIG. 19E). Comparing pre- with post-treatment, a significant reductionin MRSA biofilm burden was noted at 10 minutes of treatment with 0.5-1.0mg/mL of Lf Qi6 (FIG. 13).

Example 4—Lf Qi6 Selectively Enhances S. epidermidis while Reducing MRSAIn Vitro and on Ex Vivo Skin

The preliminary studies on biofilm modulating characteristics of Lf Qi6were studied in a 6-well pathogen-commensal co-culture model using MRSAand S. epidermidis. Results indicate that Lf Qi6 applied at 0.5-1.0mg/mL reduces MRSA biofilm pathogen burden while promoting commensalbacteria (FIG. 14A), with similar results in an ex vivo humanorganotypic culture system (FIG. 14B).

Example 5—Lf Qi6 Upregulates the Skin Barrier Homeostatic Protein,Filaggrin, at RNA Expression and at Protein Level, and SignificantlyIncreases Filaggrin Immunohistochemical Staining in an Ex Vivo HumanOrganotypic Culture System

Filaggrin immunohistochemistry shows significantly increased stainingfor filaggrin protein on the epidermis of samples from the ex vivo humanorganotypic culture system treated with 0.5-1 mg/ml application of LfQi6 for a 6-hour period (FIGS. 15A-D). Lf Qi6-treated ex-vivo skin showsan approximately 3-fold increase in filaggrin protein using ELISA (FIG.15E), as well as an approximately 250-fold increase in RNA expressionusing quantitative PCR (FIG. 15F).

Example 6—Lf Qi6 Increases Expression of the Host Innate Immune DefensePeptides, Human Beta Defensin-1, Human Beta Defensing-2, and Human BetaDefensin-3

FIG. 18 compares the increases in the expression of human beta defensins1, 2, and 3 in the presence of Lf Qi6 biofilm culture at variousconcentrations, namely 0%, 0.03%, and 0.10%, respectively, in an HEKcell model processed by ELISA (FIG. 18). The results demonstrated adose-dependent effect on the expression of the aforementioned betadefensins as well as differences in the overall effect of Lf Qi6 on eachcharacteristic beta defensing.

Furthermore, host innate immune anti-microbial peptides are integralcomponents of skin defense system. Quantitative PCR results showedapproximately 800-fold upregulation in HβD-2 (FIG. 19A) and HOD-3 (FIG.19B) in skin treated with Lf Qi6 biofilm versus the control.

Example 7—Lf Qi6 Biofilm Modulates Peroxisome Proliferator-ActivatedReceptors (PPARs)

Since Lf Qi6 increases filaggrin production and filaggrin is atranscription product of PPAR-alpha, Lf Qi6 extracts were investigatedregarding their ability to stimulate PPAR nuclear receptors. Bioactivesof Lf Qi6 biofilm extraction have been shown to increase the expressionof PPAR-beta and PPAR-gamma in keratinocytes as shown in FIG. 16. Inparticular, a small molecular size fractionated extract (<30 kDfraction, generated by differential centrifugation of the whole extractfor a particular time using a specific filter for <30 kD, e.g., AmiconUltra-15, 30K NMWL, cat# UFC 903096; extract centrifuged @ 4,000 rpm×45minutes) appeared to have more potent activity than unfractionated LfQi601 extracts.

Example 8—Lf Qi6 Biofilm can be Used as an Active Ingredient in aVariety of Moisturizing Skin-Care Products

Lf Qi6 biofilm extracts can be used as an active ingredient in a varietyof products aimed to improve the physical and biochemical functionalproperties of skin barrier. For example, Lf Qi6 biofilm extracts can beused to reduce transepidermal water loss and, in turn, improves skinhydration. The following exemplary formulations are directed to avariety of skin-care products including face lotion, eye cream, serum,and face wash. Each ingredient is presented in percent composition withits trade/brand name and international nomenclature of cosmeticingredients (INCI) name, respectively. Typical properties and methods ofpreparing each formulation are also included.

TABLE 2 Face lotion formulation comprising Lf Qi6 bioextractsTrade/Brand name INCI name Preferred Supplier % PHASE-A (OIL) Cetiol CCDicaprylyl carbomate BASF Care Creations 0.5 Cetiol OE Dicaprylyl ether″ 1.5 Olivem 1000 Cetearyl olivate sorbitan olivate Hall Star Italica1.5 Olivem Vs Feel Cetearyl alcohol, cetyl palmitate, Hall Star Italica1.5 sorbitan palmitate, sorbitan oleate Clariant Personal Care 1.5Vegetal squalane Squalane Evonik Personal care Ceramides III CeramidesOrganic Creations Inc 0.05 Kokum butter Garcinia indica butter DrAdorable 0.4 Black currant oil Ribes nigrum oil Dr Adorable 0.5 Chiaseed oil Salvia hispanica seed oil Mountain rose herbs 0.5 Borage seedoil Borago officianalis seed oil Mountain rose herbs 0.5 Eveningprimrose oil Oenothera biennis oil Extract & Ingredients 0.5 Pomegranateoil Punica granatum oil Ltd 0.5 Fractionated coconut oil Cocus nuciferaoil Dr Adorable 1.0 Lanette 22 Behenyl alcohol BASF Care Creations 0.35Vitamin E Tocopheryl acetate DSM specialty 0.05 Lexfeel N5 Diheptylsuccinate (and) capryloyl products glycerin/sebacic acid copolymerInolex Personal Care 1.5 Tegosoft P Isopropyl Palmitate Evonik Personalcare 1.5 Tegocare 450 Polyglyceryl-3 Methylglucose Distearate EvonikPersonal care 2.0 PHASE-B (WATER) Distilled Water Aqua — 79.2 Softxanthan gum Soft xanthan gum CP Kelco or lotion- 0.28 crafter Glycerin(vegetable) Glycerin Natures Answer 3.0 Aloe vera 1x Aloe Barbadensisext Making Cosmetics Inc 1.0 Xylitol Xylitol ″ 0.1 Vitamin b3Niacinamide ″ 0.5 Vitamin b5 Panthenol DSM 0.5 Citric acid — DSM 0.01PRESERVATIVES Geogard Ultra Gluconolactone and Sodium Benzoate Lonza 0.4Geogard 221 Dehydroxyacetic & Benzoic acid Lonza 0.4 ACTIVES Qi6(Lactobacillus Proprietary active Quorum Innovations 0.025 ferment) NMFProprietary active Quorum Innovations 0.2 FRAGRANCE MIX Frangipani oilPlumeria alba essential oil Eden Gardens 0.5 Jasmine oil Jasminumgrandiflorum essential oil ″ Sandal wood oil Santalum album essentialoil ″

The formulation of the natural moisturizing factor (NMF) is provided inTable 4. The face lotion was prepared using the following procedures.The oil phase (except the cermaides) was warmed to 75° C. with constantstirring. The aqueous phase was warmed to 70° C. At an optimaltemperature, e.g. 70° C., the oil phase was slowly added to the aqueousphase and the mixture was allowed to emulsify, with constant stirring,at 70-75° C. for 10 minutes. The mixture was subsequently cooled, whilestirring, to around 45-50° C. The ceramides, preservatives, actives, andfragrance mix were added to the mixture and stirred for another 10minutes. The mixture was then further mixed with a hand-held blender forapproximately 2-4 minutes, followed by vigorous mixing in a vacuum mixerfor 4 minutes. The resulting lotion blend was cooled to room temperatureand packaged into bottles. The typical properties of the face lotion arepresented in the following table.

TABLE 3 Typical properties of the face lotion. Appearance White liquidemulsion, readily flows Brookfiled viscosity at Medium viscosity at20,000 cps (LV-3 spindle) 25° C. pH at 25° C. 4.5-5.0 Storage stability1 month (4° C., 25° C. at 45° C.) Prospective fragrance Natural earthy

TABLE 4 The formulation of NMFx ™. Chemical gms/85.9 gms Free AminoAcids Serine 18.2 Arginine 9.1 Glutamic acid 3.2 Tyrosine 2.3 Alanine6.6 Sugars Xylitol 4.5 Sucrose 4.0 Bases Creatine monohydrate 1.0Glucosamine 0.5 Acids Pyrrolidone carboxylic acid 12 Hyaluronic acid 1.0Minerals Sodium chloride 5.0 Sodium lactate 5.0 Potassium citrate 0.5Calcium chloride 1.5 Magnesium chloride 1.5 Actives L-theonine 5Ceramides 5

TABLE 5 The formulation of an eye cream comprising Lf Qi6 bioextracts.Trade/Brand name INCI name Preferred Supplier (%) PHASE-A (OIL) CetiolCC Dicaprylyl carbomate BASF Care Creations 0.5 Cetiol OE Dicaprylylether ″ 1.5 Olivem 1000 Cetearyl olivate sorbitan olivate Hall StarItalica 1.65 Olivem Vs Feel Cetearyl alcohol, cetyl palmitate, Hall StarItalica 1.65 sorbitan palmitate, sorbitan oleate Olive squalane SqualaneClariant Personal Care 1.5 Ceramides III Ceramides Evonik Personal care0.05 Kokum butter Garcinia indica butter Organic Creations Inc 0.65Black currant oil Ribes nigrum oil Dr Adorable 0.5 Chia seed oil Salviahispanica seed oil Dr Adorable 0.5 Borage seed oil Borago officianalisseed oil Mountain rose herbs 0.5 Evening primrose oil Oenothera biennisoil Mountain rose herbs 0.5 Pomegranate oil Punica granatum oil Extract& Ingredients 0.5 Fractionated coconut oil Cocus nucifera oil Ltd 1.0Lanette 22 Behenyl alcohol Dr Adorable 0.35 Vitamin E Tocopheryl acetateBASF Care Creations 0.05 Lexfeel N5 Diheptyl succinate (and) capryloylDSM specialty products 1.7 glycerin/sebacic acid copolymer InolexPersonal Care Tegosoft P Isopropyl Palmitate 1.6 Tegocare 450Polyglyceryl-3 Methylglucose Evonik Personal care 2.0 Distearate EvonikPersonal care PHASE-B (WATER) Distilled Water Aqua 78.2 Soft xanthan gumSoft xanthan gum — 0.29 Glycerin (vegetable) Glycerin CP Kelco or 3.0Aloe vera 1x Aloe Barbadensis ext lotioncrafter 1.0 Xylitol XylitolNatures Answer 0.1 Vitamin b3 Niacinamide Making Cosmetics Inc 0.5Vitamin b5 Panthenol ″ 0.5 Citric acid (pH) — DSM 0.01 PRESERVATIVESGeogard Ultra Gluconolactone and Sodium DSMLonza 0.4 Benzoate Geogard221 Dehydroxyacetic & Benzoic acid Lonza 0.4 ACTIVES Qi6 (LactobacillusProprietary active Quorum Innovations 0.05 ferment) NMF Proprietaryactive Quorum Innovations 0.20 FRAGRANCE MIX Lavender oil Lavandulaangustifolio essential oil Eden Gardens 0.4

The eye cream was prepared using the following procedures. The oil phase(except the cermaides) was warmed to 75° C. with constant stirring. Theaqueous phase was warmed to 70° C. At an optimal temperature, e.g. 70°C., the oil phase was slowly added to the aqueous phase and the mixturewas allowed to emulsify, with constant stirring, at 70-75° C. for 10minutes. The mixture was subsequently cooled, while stirring, to around45-50° C. The ceramides, preservatives, actives, and fragrance mix wereadded to the mixture and stirred for another 10 minutes. The mixture wasthen further mixed with a hand-held blender for approximately 2-4minutes, followed by vigorous mixing in a vacuum mixer for 4 minutes.The resulting lotion blend was cooled to room temperature and packagedinto bottles. The typical properties of the eye cream are presented inthe following table.

TABLE 6 Typical properties of the eye cream. Appearance White liquidemulsion, readily flows Brookfiled viscosity at Medium viscosity at30,000 cps (LV-3 spindle) 25° C. pH at 25° C. 4.5-5.0 Storage stability1 month (4° C., 25° C. at 45° C.) Prospective fragrance Natural earthy

TABLE 7 The formulation of a serum comprising the Lf Qi6 bioextracts. %Ingredients Water 81.6 Aloe vera (10x) 2.0 Citric acid 0.01 Glycerin(Vegetable) 2.0 Xylitol 0.5 Green Tea Extract 0.5 Golden seal extract0.5 Hyaluronic acid BT (DSM) 0.5 Thickner (Structure cell 12000)cellulose based) 0.15 Phytoterra mate (Lonza) 0.1 Panthenol (DSM) 1.0Niacinamide (DSM) 1.0 Allantoin (Ashland) 0.05 L-Theanine (BulkNaturals) 0.1 Geogard Ultra (Lonza) 0.4 Geogard 221 (Lonza) 0.4 VitaminE 0.1 FRAGRANCES Jasmine oil (Jasminium grandiflorum) 0.6 Frankincense(Boswellia carterii) Lavender (Lavandula angustifolio) ACTIVES Qi6(Lactobacillus ferment) 0.025 NMF (moisturizing complex, minerals) 0.05

The serum was prepared by mixing the ingredients listed in Table 7, inthe order presented therein, with gentle stirring to allow thehyaluronic acid to fully hydrate and be incorporated in the mixture. ThepH was adjusted to approximately 4.5-5.0 using citric acid. The typicalproperties of the serum are listed in Table 8.

TABLE 8 Typical properties of the serum. Appearance Light brown liquidBrookfiled viscosity at Low viscosity at 1500-2000 cps (LV-3 spindle)25° C. pH at 25° C. 4.5-5.0 Storage stability 1 month (4° C., 25° C. at45° C.) Prospective fragrance Natural earthy

TABLE 9 The formulation of a face-wash comprising the Lf Qi6 bioextract.Trade Name INCI Name (or generic name) % Aqua — 30.0 Miranol Ultra L 32(solvay) Sodium lauroampho acetate 25.0 (Solvay) MiraCare Plaisant(solvay) Sodium cocoyl Isethionate + 25.0 Sodium lauramphoacetate 10.0Planteron 2000 (BASF) Decyl glucosides 2.5 Glycerin Vegetable (Jedwards)2.0 Amaranth S Sodium cocyl hydrolyzed 0.5 Amaranth ext (Lonza) XylitolXylitol (Spectrum chemicals) 0.5 Panthenol (DSM) — 0.5 Niacinamide (DSM)— 3.0 Aloe vera Aloe vera 0.45 Geogard Ultra Gluconolactone + Sodium0.45 Benzoate Geogard 221 Dehydroxyacetic & Benzoic acid 0.76 Citricacid (pH Adjuster) — Qi6 (Lactobacillus ferment) Lactobacillus ferment0.001 NMF Qi-NMF Moisturizing complex 0.075 Fragrance mix Lavandulaangustifolio, Mentha 0.45 (Lavender oil:Spearmint spicata 6:1)

The face-wash was prepared using the following procedures. Water wasfirst added into a mixing vessel and the vessel was turned on to lowspeed (100-150 rpm). Xylitol, panthenol, niacinamide, NMF, Geogard Ultrawas sequentially added and dissolved in water. Geogard 221 and Lf Qi6bioextracts were subsequently added to the mixture. MiraCare Plaisantwas then added to the mixture and stirred until the mixture turnedclear. Miranol Ultra 32 was added to the mixture and stirred until themixture turned clear, which was followed by the addition of Plaanteron2000 to the mixture. The mixture was then stirred until it turned clearand its pH was subsequently adjusted using citric acid. The typicalproperties of the face-wash are listed in Table 10 below.

TABLE 10 Typical properties of the face-wash. Appearance Clear viscousliquid Brookfiled viscosity at 25° C. 6000-8000 cps (LV-3 spindle, 12rpm) pH at 25° C. 6.5-7.0 Storage stability 1 month (4° C., 25° C. at45° C.) Prospective fragrance Natural minty

Example 9—Treatment of Eosinophilic Gastrointestinal Diseases

Eosinophilic gastrointestinal diseases have risen in incidencesignificantly in the past few decades. Of these diseases, eosinophilicesophagitis is the most common. Current treatments are difficult and/orinvolve systemic steroid exposure. For instance, an elemental diet freeof food allergen such as milk, wheat, seafood and soy is effective butburdensome to the patient. Therefore, oral slurries of topical steroidsare often used. This has the advantage of delivering the therapeuticcomponent directly to the inflamed tissues. Despite its efficacy, oralsteroids can have potential side effects, such as immunosuppression,growth velocity reduction, and adverse bone density effects. There is animmediate need for effective, non-burdensome, steroid-free treatmentsfor eosinophilic gastrointestinal disease. Mucoadhesives areadvantageously used to increase local tissue exposure to the activecomponents of the formulation. Here we describe a pharmaceuticalformulation of a stable, high-viscosity orally-administered gelcontaining a Lactobacillus extract. A steroid such as budesonide (e.g.,0.01% of total formulation) may be added if desired.

Lactobacillus extract 0.001-0.5 g Parabens 0.5 g Propylene glycol 5 g*Lutrol F127 20 g Water 75 g Total: 100 gDissolve Lutrol F127 and parabens in water heated to 80 degrees C.Add propylene glycol and lactobacillus extract.Keep heated until a clear colorless gel is obtained. *Alternativemucoadhesives may be used, such as carboxymethyl cellulose,hydroxypropyl methyl cellulose, or hydrodroxypropyl cellulose.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and the scope of the appended claims.

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What is claimed is:
 1. A method of inhibiting microbial growth on asurface, wherein the method comprises applying to the surface amicrobial growth-inhibiting amount of a composition comprising abioactive L. fermentum bacterial strain grown as biofilm, and/orcomprising a bioactive extract of said biofilm, wherein the bacterialstrain is Lactobacillus fermentum Qi6 having Accession No. PTA-122195.2. The method, according to claim 1, wherein the surface is skin.
 3. Themethod, according to claim 2, wherein skin barrier function is enhanced.4. The method, according to claim 2, wherein the composition acts as anagonist of PPAR.
 5. The method, according to claim 1, wherein thecomposition comprises alcohol.
 6. The method, according to claim 1,wherein the method promotes the growth of commensal bacteria.
 7. Themethod according to claim 6, wherein the commensal bacteria areStaphylococcus epidermidis.
 8. The method, according to claim 1, whereinthe surface is an inanimate surface.
 9. The method, according to claim1, wherein the composition comprises a bioactive extract of the L.fermentum biofilm.
 10. The method, according to claim 1, used to inhibitthe growth of methicillin-resistant Staphylococcus aureus (MRSA).
 11. Abiologically pure culture of Lactobacillus fermentum Qi6 havingAccession No. PTA-122195.
 12. A composition comprising Lactobacillusfermentum Qi6 having Accession No. PTA-122195, and a carrier.
 13. Thecomposition, according to claim 12, comprising an edible material.